Ex vivo detection of SARS-CoV-2-specific CD8+ T cells: rapid induction, prolonged contraction, and formation of functional memory

  1. Isabel Schulien
  2. Janine Kemming
  3. Valerie Oberhardt
  4. Katharina Wild
  5. Lea M. Seidel
  6. Saskia Killmer
  7. Sagar
  8. Franziska Daul
  9. Marilyn Salvat Lago
  10. Annegrit Decker
  11. Hendrik Luxenburger
  12. Benedikt Binder
  13. Dominik Bettinger
  14. Oezlem Sogukpinar
  15. Siegbert Rieg
  16. Marcus Panning
  17. Daniela Huzly
  18. Martin Schwemmle
  19. Georg Kochs
  20. Cornelius F. Waller
  21. Alexandra Nieters
  22. Daniel Duerschmied
  23. Florian Emmerich
  24. Henrik Mei
  25. Axel Schulz
  26. Sian Llewellyn-Lacey
  27. David A. Price
  28. Tobias Boettler
  29. Bertram Bengsch
  30. Robert Thimme
  31. Maike Hofmann
  32. Christoph Neumann-Haefelin

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Abstract

CD8+ T cells are critical for the elimination and long-lasting protection of many viral infections, but their role in the current SARS-CoV-2 pandemic is unclear. Emerging data indicates that SARS-CoV-2-specific CD8+ T cells are detectable in the majority of individuals recovering from SARS-CoV-2 infection. However, optimal virus-specific epitopes, the role of pre-existing heterologous immunity as well as their kinetics and differentiation program during disease control have not been defined in detail. Here, we show that both pre-existing and newly induced SARS-CoV-2-specific CD8+ T-cell responses are potentially important determinants of immune protection in mild SARS-CoV-2 infection. In particular, our results can be summarized as follows: First, immunodominant SARS-CoV-2-specific CD8+ T-cell epitopes are targeted in the majority of individuals with convalescent SARS-CoV-2 infection. Second, MHC class I tetramer analyses revealed the emergence of phenotypically diverse and functionally competent pre-existing and newly induced SARS-CoV-2-specific memory CD8+ T cells that showed similar characteristics compared to influenza-specific CD8+ T cells. Third, SARS-CoV-2-specific CD8+ T-cell responses are more robustly detectable than antibodies against the SARS-CoV-2-spike protein. This was confirmed in a longitudinal analysis of acute-resolving infection that demonstrated rapid induction of the SARS-CoV-2-specific CD8+ T cells within a week followed by a prolonged contraction phase that outlasted the waning humoral immune response indicating that CD8+ T-cell responses might serve as a more precise correlate of antiviral immunity than antibody measurements after convalescence. Collectively, these data provide new insights into the fine specificity, heterogeneity, and dynamics of SARS-CoV-2-specific memory CD8+ T cells, potentially informing the rational development of a protective vaccine against SARS-CoV-2.

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  1. ScreenIT

    SciScore for 10.1101/2020.08.13.249433: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board StatementConsent: Written informed consent was obtained from all participants and the study was conducted according to federal guidelines, local ethics committee regulations (Albert-Ludwigs-Universität, Freiburg, Germany; vote #: 322/20) and the Declaration of Helsinki (1975).
    IRB: Written informed consent was obtained from all participants and the study was conducted according to federal guidelines, local ethics committee regulations (Albert-Ludwigs-Universität, Freiburg, Germany; vote #: 322/20) and the Declaration of Helsinki (1975).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Multiparametric flow cytometry: The following antibodies were used for multiparametric flow cytometry: anti-CCR7-PE-CF594 (150503, 1:50), anti-CCR7-BUV395 (3D12, 1:50), anti-CCR7-BV421 (150503, 1:33), anti-CD4-BV786 (L200, 1:200), anti-CD8-BUV496 (SK1, 1:100), anti-CD8-BUV510 (SK1, 1:100), anti-CD8-APC (SK-1, 1:200), anti-CD27-BV605 (L128, 1:200), anti-CD28-BV421 (CD28.2, 1:100), anti-CD28-BV711 (CD28.2, 1:100), anti-CD45RA-BV786 (HI100, 1:800), anti-CD45RA-BUV737 (HI100, 1:200), anti-CD69-BUV395 (FN50, 1:50), anti-CD107a-APC (H4A3, 1:100), anti-CD127-BV510 (HIL-7R-M21, 1:25), anti-EOMES-PerCP-eF710 (WD1928, 1:50), anti-IFN-γ-FITC (25723.11, 1:8), anti-IL-21-PE (3A3-N2.1, 1:25), anti-PD-1-BV786 (EH12.1, 1:33), anti-TNF-PE-Cy7 (Mab11, 1:400
    anti-CCR7-PE-CF594 ( 150503
    suggested: None
    anti-CCR7-BUV395
    suggested: None
    3D12
    suggested: (LSBio (LifeSpan Cat# LS-C52290-200, RRID:AB_1275093)
    anti-CCR7-BV421
    suggested: None
    anti-CD4-BV786
    suggested: None
    anti-CD8-BUV496
    suggested: None
    anti-CD8-BUV510
    suggested: None
    SK1
    suggested: (Millipore Cat# AB9772-200UL, RRID:AB_672977)
    anti-CD8-APC ( SK-1
    suggested: None
    anti-CD27-BV605 ( L128
    suggested: None
    anti-CD28-BV421 ( CD28.2
    suggested: None
    anti-CD28-BV711 ( CD28.2
    suggested: None
    anti-CD45RA-BV786
    suggested: None
    HI100
    suggested: (Rockland Cat# 200-302-N70, RRID:AB_2611455)
    anti-CD45RA-BUV737 ( HI100
    suggested: None
    anti-CD69-BUV395
    suggested: None
    FN50
    suggested: None
    anti-CD107a-APC
    suggested: None
    anti-CD127-BV510
    suggested: None
    HIL-7R-M21
    suggested: None
    anti-EOMES-PerCP-eF710 ( WD1928
    suggested: None
    anti-IFN-γ-FITC
    suggested: None
    anti-IL-21-PE ( 3A3-N2.1
    suggested: None
    anti-PD-1-BV786
    suggested: None
    anti-TNF-PE-Cy7
    suggested: None
    Mab11
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Analysis of panel 1 (transcription factors) included the markers CD45RA, CCR7, CD27, CD28, BCL-2, TCF-1, CD69, CD38, PD-1, EOMES, T-BET and TOX.
    BCL-2
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    HLA class I easYmers® (immunAware, Copenhagen, Denmark) were loaded with peptide according to manufacturer’s instructions (A*01/ORF3a207-215, A*01/ORF1ab4163-4172, A*02/ORF3a139-147, B*07/N105-113) or ordered as peptide-loaded monomers (B*44:03/N322-330, B*44:03/ORF1ab3946-3954).
    B*44:03/N322-330
    suggested: None
    Software and Algorithms
    SentencesResources
    Sequence Alignment: Sequence homology analyses were performed in Geneious Prime 2020.0.3 (https://www.geneious.com/) using Clustal Omega 1.2.2 alignment with default settings12.
    https://www.geneious.com/
    suggested: (Geneious, RRID:SCR_010519)
    Clustal Omega
    suggested: (Clustal Omega, RRID:SCR_001591)
    Reference genomes of human coronaviruses were downloaded from NCBI database 229E (
    NCBI
    suggested: (NCBI, RRID:SCR_006472)
    Dimensionality reduction of multiparametric flow cytometry data: The visualization of multiparametric flow cytometry data was done with R version 4.0.2 using the Bioconductor (version: Release (3.11)
    Bioconductor
    suggested: (Bioconductor, RRID:SCR_006442)
    CATALYST package (Crowell H, Zanotelli V, Chevrier S, Robinson M (2020).
    CATALYST
    suggested: (CATALYST, RRID:SCR_017127)
    For analysis of mass cytometric data samples were first gated on Iridium intercalator positive, live, single CD45+CD3+CD8+ T cells using FlowJo (v10.6).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Further analysis and heatmap visualization was performed using R (v4.0) (https://www.r-project.org).
    https://www.r-project.org
    suggested: (R Project for Statistical Computing, RRID:SCR_001905)
    Statistics: Statistical analysis was performed with GraphPad Prism 8 (USA).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    Clearly, our approach to define optimal CD8+ T-cell epitopes based on a in silico prediction has the limitation that it does not completely cover the entire viral genome as it is the case in studies that have used overlapping peptides3, 7, 9. However, an advantage of this approach is the definition of exact, single and optimal CD8+ T-cell epitopes including HLA restriction. With this, our data revealed that there is no clear dominance of HLA-A or B-restricted epitopes that are targeted by SARS-CoV-2-specific CD8+ T cells indicating an evenly broad and robust induction of an antiviral CD8+ T-cell response among individuals. Importantly, the definition of optimal epitopes also allowed a comparative ex vivo detection and characterization of SARS-CoV-2-specific CD8+ T cells after peptide-loaded MHC I tetramer-based enrichment. The highest frequency ex vivo was detectable for B*07/N105-113-specific CD8+ T cells which was also in agreement with their strong peptide-specific expansion. Indeed, B*07/N105-113-specific CD8+ T cells were similarly frequent as A*02/Flu-M158-66-specific CD8+ T cells. Since the sequence homology of the B*07/N105-113 epitope among the corona viruses including “common cold” corona viruses is high and since we also identified pre-existing B*07/N105-113-specific CD8+ T cells in historic controls, the higher frequency of these CD8+ T cells in SARS-CoV-2 convalescent individuals most probably reflects heterologous boosting. Of note, in the study by Peng et al.7,...

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 24, 25, 26 and 27. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.1101/2020.08.13.249433v1 on bioRxiv